Yeast aquaglyceroporins use the transmembrane core to restrict glycerol transport

Abstract

Aquaglyceroporins are transmembrane proteins belonging to the family of aquaporins, which facilitate the passage of specific uncharged solutes across membranes of cells. The yeast aquaglyceroporin Fps1 is important for osmoadaptation by regulating intracellular glycerol levels during changes in external osmolarity. Upon high osmolarity conditions, yeast accumulates glycerol by increased production of the osmolyte and by restricting glycerol efflux through Fps1. The extended cytosolic termini of Fps1 contain short domains that are important for regulating glycerol flux through the channel. Here we show that the transmembrane core of the protein plays an equally important role. The evidence is based on results from an intragenic suppressor mutation screen and domain swapping between the regulated variant of Fps1 from Saccharomyces cerevisiae and the hyperactive Fps1 ortholog from Ashbya gossypii. This suggests a novel mechanism for regulation of glycerol flux in yeast, where the termini alone are not sufficient to restrict Fps1 transport. We propose that glycerol flux through the channel is regulated by interplay between the transmembrane helices and the termini. This mechanism enables yeast cells to fine-tune intracellular glycerol levels at a wide range of extracellular osmolarities.

FIGURE 1.

AgFps1 mediates glycerol flux in A. gossypii. A, growth of wild type and Agfps1Δ at 18 h after the addition of YNB supplemented with 2% glucose or 2% glycerol to spores. B, influx of glycerol in wild type and Agfps1Δ grown in YNB supplemented with 2% glycerol as a measure of AgFps1 channel activity. Measurements were performed at least three times, and error bars indicate S.D. C, growth of wild type and Agfps1Δ at 18 h after the addition of YPD with or without arsenite to spores. A and C, filamentous growth is indicated by white arrows, and nongerminated, needle-shaped spores are shown by black arrows. More needle-shaped spores indicate less efficient germination and growth.

FIGURE 2.

Characterization of AgFps1 by heterologous expression in S. cerevisiae. Growth phenotypes of the Scfps1Δ transformed with empty plasmid or plasmids containing FPS1 alleles were determined. A, Scfps1Δ mutants carrying empty plasmid, ScFPS1, or AgFPS1 were shifted from high (1 m sorbitol) to low (no sorbitol) osmolarity. A higher proportion of cells surviving the hypo-osmotic shock when compared with cells with empty plasmid indicates a functional Fps1 channel. B, growth phenotype of the Scfps1Δ mutant carrying empty plasmid, ScFPS1, AgFPS1, or ScFPS1-N228A in the absence (control) or presence of 0.8 m NaCl. Failure to grow on salt medium indicates expression of a hyperactive channel.

FIGURE 3.

Determination of the role of the transmembrane core in controlling Fps1 activity. A, sketch of ScFPS1 and AgFPS1 chimeras. Different combinations of the N and C termini and the transmembrane cores of ScFPS1 and AgFPS1 were fused together and expressed under the control of the ScFPS1 promoter in the YEp181myc plasmid. The numbers refer to the first and the last amino acids of each part of the chimera. For further details, see supplemental Fig. S1. B, growth phenotype of the Scfps1Δ mutant carrying empty plasmid or FPS1 alleles, pregrown on 1 m sorbitol and shifted to low osmolarity (no sorbitol). Increased survival rate when compared with cells with empty plasmid indicates functional channels. C and D, growth curves, monitored in a Bioscreen automatic reader, of Scfps1Δ cells expressing wild type and chimera proteins cultured in YNB medium (C) and in YNB medium containing 1 m sorbitol (D). Expression of hyperactive channels affects growth in high osmolarity medium. OD indicates optical density.

FIGURE 4.

Identification of a transmembrane core residue important for channel activity. A, growth phenotypes of the Scfps1Δ transformed with empty plasmid or plasmids containing ScFPS1 alleles. Increased survival rate when compared with cells with empty plasmid indicates functional channels. Poor growth on NaCl in the Scfps1Δ mutant and growth on xylitol in the gpd1Δgpd2Δ mutant indicate hyperactive ScFps1 channels. B, cells were stressed with 0.8 m NaCl to allow accumulation of internally produced glycerol, and intracellular glycerol concentrations/A₆₀₀ over time was determined. C, influx of glycerol into Scfps1Δ mutant expressing different ScFPS1 alleles as a measure of ScFps1 channel activity. High glycerol influx indicates a hyperactive channel. Measurements were performed at least three times, and error bars indicate S.D. OD indicates optical density.

FIGURE 5.

Mutation of a conserved glycine in TM6 negatively affects glycerol transmembrane flux. A, structural model of the ScFps1 transmembrane core using the E. coli aquaglyceroporin GlpF structure as template. B, alignment of the TM6 from ScFps1, AgFps1, and other aquaporins or aquaglyceroporins from E. coli, human, and Arabidopsis thaliana. C, growth phenotypes of the Scfps1Δ transformed with empty plasmid or plasmids containing ScFPS1 alleles. Increased survival rate when compared with cells with empty plasmid indicates functional channels. Poor growth on NaCl in the Scfps1Δ mutant and growth on xylitol in the gpd1Δgpd2Δ mutant indicate hyperactive ScFps1 channels. D, influx of glycerol into Scfps1Δ mutant expressing AgFPS1 and AgFps1-G382S as a measure for AgFps1 channel activity. High influx indicates a hyperactive channel. Measurements were performed at least three times and error bars indicate S.D.